System for collecting condensed liquid samples containing exhaled aerosol particles - Patents.com

Abstract

To provide methods and devices for EBA and EBC analysis to provide a reliable diagnostic result, including tuberculosis diagnosis, in less than 30 minutes, and preferably in less than 20 minutes, to enable point of care health services and minimize disease transmission using Active Case Finding.SOLUTION: Disclosed are methods and devices for analyzing exhaled aerosols and exhaled breath condensate using various diagnostic tools that enable rapid, low cost and autonomous point of care assays for several diseases including respiratory tract diseases. Also disclosed are methods and devices for analyzing exhaled aerosols and exhaled breath condensate for tuberculosis diagnosis using mass spectrometry, including MALDI-MS. The disclosed systems and methods provide for a diagnostic test result in less than about 20 minutes and autonomous operation with minimal human intervention.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] This application relates to a system for collecting condensed liquid samples containing exhaled breath aerosol (EBA) particles. [Background technology]

[0002] The present disclosure relates to methods and apparatus for analyzing exhaled breath aerosol and exhaled breath condensate using various diagnostic tools to enable rapid, low-cost, autonomous point-of-care assays for several diseases, including respiratory diseases. More specifically, but not by way of limitation, the present disclosure relates to methods and apparatus for analyzing exhaled breath aerosol and exhaled breath condensate for tuberculosis diagnosis using mass spectrometry, including MALDI-MS.

[0003] Tuberculosis (TB) has surpassed HIV / AIDS as a global killer, with over 4000 deaths every day (Patterson, B., et al., 2018). Incidence declines remain insufficient at a reported 1.5% per year, making it unlikely that treatment alone will significantly reduce the burden of disease. In communities where HIV is highly prevalent, Mycobacterium tuberculosis (Mtb) genotyping studies have found that recent infection, rather than reactivation, accounts for the majority (54%) of TB cases. The physical process of TB transmission is still poorly understood, and the application of new technologies to elucidate key events in the generation, release, and inhalation of infectious aerosols has been slow. Empirical studies to characterize airborne infectious particles are sparse. Two major problems plaguing investigations are the low concentrations of naturally produced Mtb particles and the complexity of environmental and patient-derived bacterial and fungal contamination of airborne samples. Nevertheless, there are numerous attempts at airborne detection. A 2004 proof-of-concept study and subsequent feasibility study in Uganda sampled cough-generated aerosols from patients with pulmonary tuberculosis. Coughing directly into a sampling chamber equipped with two workable cascade impactors yielded positive cultures from more than a quarter of participants, despite having received chemotherapy for 1–6 days. A follow-up study using the same device found that participants with higher aerosol bacterial loads may be associated with higher household infection rates and disease progression, and quantitative airborne sampling may serve as a clinically relevant measure of infectivity. Thus, interruption of transmission may have a rapid and measurable impact on tuberculosis incidence.

[0004] The best way to control the transmission of tuberculosis is to rapidly identify and treat active TB cases (Wood, RC, et al., 2015). The diagnosis of pulmonary TB is usually made by microbiological, microscopic, or molecular analysis of the patient's sputum. The "gold standard" test for TB infection in most of the developing world is smear culture based on a sputum sample. The sample is smeared on a culture plate, a stain specific for Mtb is added, and the stained cells are counted using a microscope. If the concentration of cells in the smear is higher than a set threshold, the sample is classified as positive. If the TB count is below this threshold, it is classified as a negative value. The diagnosis may take several hours. The need for sputum as a diagnostic sample is a limiting factor due to the challenges of collecting sputum from patients and its complex composition. The viscosity of the material limits the sensitivity of the test, increases the heterogeneity between samples, and increases the costs and labor associated with the test. Furthermore, the production of sputum (which requires coughing) is an occupational hazard for health care workers. Sputum has several drawbacks as a sample medium. First, only about 50% of patients can provide a good sputum sample. For example, children under about 8 years of age often cannot produce a sample on demand because they usually do not have developed the ability to "cough up" sputum from the back of their throat. Elderly and sick people may not have the strength to cough up sputum. Others may simply not have sputum in their throat. Thus, diagnostic methods based on sputum analysis may not provide a diagnosis in as many as 50% of patients who provide a sputum sample. Sputum is also useless as a diagnostic sample if collected 1-2 days after being treated with antibiotics, as it does not represent deep lung pathology. Within a few days after the start of treatment, the number of live Mtb in sputum is significantly reduced. Urine and blood have been proposed as sample media for the diagnosis of TB infection. Blood is highly invasive and there are high costs associated with handling blood samples that are often HIV positive, because in some parts of the world, many TB patients are also co-infected with HIV, and moreover, patients with active TB may have fewer circulating TB cells in their blood.Urine-based diagnostics have also been proposed, but these tests look for biomarkers of disease other than viable TB bacteria and have not been validated for widespread clinical use.

[0005] Easier, safer, and more homogeneous samples to collect and handle would simplify TB diagnosis. Exhaled breath contains aerosols ("EBA") and vapors that can be collected noninvasively and characterized to elucidate physiological and pathological processes in the lung (Hunt, 2002). To capture exhaled breath for analysis, it passes through a condenser to produce a liquid accumulation called exhaled breath condensate ("EBC"). Although EBC is derived primarily from water vapor, EBC contains dissolved nonvolatile compounds, including cytokines, lipids, surfactants, ions, oxidation products, adenosine, histamine, acetylcholine, and serotonin. In addition, EBC traps potentially volatile water-soluble compounds such as ammonia, hydrogen peroxide, and ethanol, as well as other volatile organic compounds. The pH of EBC can be easily measured. EBC contains aerosolized airway lining fluid and volatile compounds, providing a noninvasive indication of ongoing biochemical and inflammatory activity in the lung. The rapidly growing interest in EBCs stems from the realization that in pulmonary disease, EBCs have measurable characteristics that can be used to distinguish infected from healthy individuals. These assays have provided evidence of airway and lung redox deviations, acid-base status, and the degree and type of inflammation in acute and chronic asthma, chronic obstructive pulmonary disease, adult respiratory disease syndrome, occupational diseases, and cystic fibrosis. Characterized by uncertain and variable dilutions, EBCs may not provide an accurate assessment of individual solute concentrations within the native airway lining fluid. However, they can provide useful information when concentrations differ substantially between health and disease or are based on the ratio of solutes detected in the sample.

[0006] Patterson et al. (2018) used a custom-built respiratory aerosol sampling chamber (RASC), a novel device designed to optimize patient-derived exhaled aerosol sampling and to isolate and accumulate respirable aerosols from a single patient. Environmental sampling detects Mtb present after a period of aging in the chamber air. Thirty-five newly diagnosed GeneXpert (Cepheid, Inc., Sunnyvale, CA) sputum-positive TB patients were sampled in approximately 1.4 m 3 The patients were monitored while confined for one hour in the RASC chamber. The GeneXpert genetic assay is based on polymerase chain reaction (PCR) and can be used to analyze samples for BT diagnosis and to indicate whether a TB sample has drug resistance genes. The GeneXpert PCR assay for TB accepts sputum samples and provides a positive or negative result in about one hour. The chamber is equipped with aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO 2 Monitoring, and cough recordings were incorporated. Microbial culture and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each bioaerosol collection device. Mtb was detected in 77% of aerosol samples, with 42% of samples being positive by mycobacterial culture and 92% being positive by ddPCR. A correlation was found between the incidence of cough and culturable bioaerosols. Mtb was detected in all viable cascade impactor stages with a peak in aerosol sizes between 2.0 and 3.5 μm. This was consistent with a median aerosol culture-positive exhaled breath of 0.09 CFU / liter and a median estimate of exhaled particulate bioaerosols of 4.5x10 7 CFU / ml. Mtb was detected in bioaerosols exhaled by the majority of untreated TB patients using the RASC chamber. Molecular detection was found to be more sensitive than Mtb culture on solid media.

[0007] Mtb can be identified in EBA by culture, ddPCR, electron microscopy, immunoassays, and cell stains (such as oramine and dmnTre). Of these, PCR and immunoassays can be rapid and specific to the species level. PCR and other genomics-based techniques can be specific to the strain level. Mass spectrometry has been shown to be specific to the strain level in cultures obtained from bacterial infections. For example, the Biotyper from Bruker Daltonics (Germany) has been shown to be able to identify up to 15,000 strains of bacteria that cause infections in humans. These techniques have been shown to be able to identify tuberculosis infections from EBA. Immunoassays for Mtb detection, such as those based on lipoarabinomannan, are also well known.

[0008] In the case of TB, people infected with TB are often diagnosed by passive case finding when an individual presents to a clinic. Active case finding (ACF) is generally considered to include other methods of reaching people suspected of having TB infection outside the primary health care system. According to the WHO, ACF is the "systematic identification of people suspected of having active TB using rapidly applicable tests, screening, or other procedures." The goal of ACF is to treat infected people earlier, shorten the average infectious period, and thereby reduce the spread of the disease. In the case of TB, by the time an individual presents to a clinic for help, he or she may have transmitted TB infection to about 10 to about 115 other people. ACF helps to reduce or prevent significant transmission of TB. Diagnostic systems and methods, such as sputum analysis and blood analysis, are not automated, do not operate autonomously, or are not rapid. Many require expensive assays that are consumable per analysis, and therefore lack general utility for active case finding, especially in underdeveloped and developing countries. As mentioned earlier, EBA analysis would be a compelling diagnostic tool for TB detection offering rapid analysis, portability, and low cost as it eliminates the need for expensive assays and consumables. McDevitt et al. (2013) report an EBA analysis device and method for influenza diagnosis. An impactor is used to remove large particles (>4 μm) from exhaled breath, followed by a wet film collector for smaller particles (<4 μm). Two size bins of collected particles were analyzed for influenza virus using reverse transcriptase polymerase chain reaction (rt-PCR), a genomics-based method. PCR technology uses biomolecular probes combined with other biomolecules, including enzymes, to amplify specific sequences of DNA if they are present in the sample. The targeted sequences are believed to be specific for the disease being identified. McDevitt et al. showed that EBA samples can be used to diagnose influenza. The disclosed device and method have several drawbacks from a practical standpoint.First, exhaled aerosol samples are collected in individual samples of several milliliters in volume, so a significant effort is required to concentrate the samples. Furthermore, the diagnostic device is not coupled or integrated with a sample collector and is not suitable for use as an ACF tool. The ability to automate the RNA assay to create an autonomous diagnostic tool for TB analysis is not clear. No method has been described to determine whether a sufficient amount of cough or exhaled aerosols was generated by a particular patient. As a result, if a sample turns out to be negative for influenza, it may be due to a false negative resulting from insufficient sample collection. It is well known that there is a large variability among humans regarding the amount of aerosolized lung fluid generated during various breathing maneuvers.

[0009] GeneXpert Ultar is a cutting-edge genomics-based point-of-care diagnostic device that uses PCR technology. It can also be integrated with EBA sample collection methods to perform ACF for tuberculosis and other respiratory diseases, but the sample collection time is too long to be practical. Patterson et al. have shown that 20–200 TB bacteria are typically produced in EBA and can be collected in a 1-hour sampling period. A minimum of 1 hour of sampling is required to use GeneXpert Ultra as a diagnostic assay. GeneXpert can be integrated with systems that sample air and analyze air samples for airborne pathogens. The BDS system (Northup Grumman, Edgewood, MD) is used to screen mail for the United States Postal Service for the bacterial spores that cause anthrax as the mail passes through distribution centers. It combines a wetted-wall cyclone with the GeneXpert PCR system to autonomously sample the air and report whether the pathogen is present. However, the GeneXpert Ultra assay has a relatively high cost per test and takes approximately 1 hour to complete the assay and provide results. In general, PCR-based diagnostics are not suitable for TB screening for ACF applications due to the long time required for sampling and analysis and the relatively high cost per test.

[0010] The time associated with a diagnostic assay is a critical parameter for field or "point-of-care" testing. By definition, ACF is an example of a field diagnostic assay because it is performed outside of the healthcare system. In the United States, point-of-care tests must provide an answer within 20 minutes. If not, the test is considered too slow and unacceptable to achieve short patient wait times. In developing countries, especially those with a history of TB epidemics, a diagnosis can be made in about an hour using GeneXpert. As mentioned earlier, this assay is expensive to implement on a "cost per test" basis and therefore has not yet been widely deployed. Due to the high cost, it is not used to screen patients who appear healthy (asymptomatic) but may be infected with TB, but is used to confirm a diagnosis that is strongly suspected based on other tests and factors.

[0011] Fennelly et al. (2004) described a TB analysis using cough aerosols and a collection chamber containing two Anderson cascade impactors using individuals known to be active patients. The individuals were asked to provide two separate 5-min bursts of vigorous coughing. Cultivation of the impacted samples took 30–60 days, making this approach not amenable to automation. A challenging aspect of EBA as a clinical sample is the relatively small sample size of the amount of exhaled particles that can be collected from exhaled breath. Furthermore, a significant portion of the collected mass is water. Molecules containing diagnostic information ("biomarkers") are present in nanoliter or picogram quantities. The aerosol collection method must then be effective in capturing the majority of the biomass in exhaled breath. Exhaled breath includes the air expelled from the lungs by various maneuvers, such as breathing, deep breathing, coughing, and sneezing. Certain types of deep-breathing maneuvers, such as forced vital capacity (FVC), can be used to measure maximum vital capacity by breathing in as far as possible and exhaling as deeply (or as deeply) as possible to maximize the volume of exhalation. Forced expiratory volume (FEV) measures the amount of air a person can exhale during a forced breath. The volume of exhaled air can be measured during 1 second (FEV1), 2 seconds (FEV2), and / or 3 seconds (FEV3) of a forced breath. Forced vital capacity (FVC) is the total volume of air exhaled during an FEV test. Forced expiratory volume and forced vital capacity are pulmonary function tests measured during spirometry. Forced expiratory volume is an important measurement of lung function.

[0012] Although studies have shown that respiratory diseases can be detected from exhaled aerosols and exhaled breath condensate, modern clinical testing for infections and diseases such as tuberculosis, influenza, and pneumonia continues to utilize sputum, blood, and nasal swabs. Breath analysis tools have not been commercialized due to a lack of methods and devices to efficiently collect and concentrate trace amounts of analytes present in exhaled breath. Furthermore, there is no standard or methodology for assessing how much breath is sufficient for a specific diagnosis. The disclosed exemplary devices and methods overcome these limitations by collecting exhaled aerosols and exhaled breath condensate into a high-flow, high-efficiency, relatively concentrated sample. Additionally, size sorting of the aerosols can be incorporated to increase the signal-to-noise ratio of certain analytes prior to collecting the analytes. The concentrated samples can then be analyzed by several methods, preferably using methods that are sensitive, rapid, and highly specific for the analytes of interest. More preferably, the analysis is rapid and near real-time. Mass spectrometry, real-time PCR, and immunoassays offer the best chance of being sensitive, specific, and near real-time.

[0013] There is a need for sample collection methods that can provide diagnostic assays that are fast, sensitive, detailed, and preferably characterized by a low cost per test, in combination with rapid diagnostic tools such as mass spectrometry ("MS"), which is faster and more reliable than sputum analysis and less invasive than blood analysis. Such systems can be used for active case finding (ACF) of TB and other pulmonary or respiratory diseases. To be effective, ACF systems must be fast and inexpensive on a "per diagnosis" basis. A low cost per test is required to screen large numbers of individuals to proactively prevent TB infection and look for the few who do have TB. Low-cost devices and methods will also be needed for point-of-care diagnostics of influenza and other pathogenic viruses, since patients with the "common cold" may be infected with rhinovirus. In some cases, respiratory infections are caused by bacterial or fungal microorganisms and may be treatable with antibiotics. In other cases, the microorganisms may be resistant to antibiotics, and diagnostic methods that can identify the resistance of the microorganism to antibiotics are preferred. A rapid EBA method is desired to distinguish between viral and bacterial infections in the respiratory tract while minimizing false negatives due to insufficient sample volume. Mass spectrometry, genomics methods including PCR, and immunoassays have the potential to be the most sensitive and specific. Mass spectrometry, particularly MALDI time-of-flight mass spectrometry (MALDI-TOFMS), has been demonstrated to be sensitive, specific, and near real-time, making it the preferred diagnostic tool for the analysis of EBA and EBC samples. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Application Publication No. 9-126958 [Patent Document 2] International Publication No. 2019 / 011750 Summary of the Invention

[0015] Disclosed herein are exemplary methods and devices for EBA and EBC analysis to provide reliable diagnostic results, including tuberculosis diagnosis, in less than 30 minutes, preferably less than 20 minutes, to enable point-of-care health services and minimize disease transmission using Active Case Finding, which are also characterized by low cost per patient and are autonomous.

[0016] An autonomous system for diagnosing respiratory diseases in an individual using exhaled breath is disclosed, the system comprising a sample collection subsystem having a sample extraction component configured to receive the face of the individual to extract exhaled breath aerosol (EBA) particles emitted from the individual during the predetermined number of breathing maneuvers into the air flow provided to an extraction component, a sample capture component fluidly connected to the sample extraction component by an interface tube and configured to separate and collect EBA particles from the exhaled breath and air as a collected sample, a sample analysis subsystem fluidly connected to the sample capture component, the sample analysis subsystem having a sample processing component for spotting a small amount of the collected sample onto a sample plate and concentrating the collected sample on the sample plate, and a diagnostic device for analyzing the sample. The EBA particles may comprise at least one of microbial, viral, metabolite biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of the respiratory disease. The flow rate of air entering the sample capture component may be between about 100 L / min and about 1000 L / min. The flow rate of air entering the sample capture component may be between about 50 L / min and about 500 L / min. A collected sample volume may be between about 100 microliters and about 1 ml. The sample capture component may further include an air pump and an impactor, where the air pump provides an air flow to transport the exhaled breath from the extraction component to the impactor, which separates the EBA particles from the exhaled breath to generate a collected sample. The impactor may include at least one of a cyclone, a wetted-wall cyclone, one or more wetted film impactors, and an impinger. The system may further include at least one virtual impaction stage located upstream of the impactor.The sample extraction component may include at least one of a cone-shaped device, a shroud, a CPR rescue mask, a CPAP mask, a respirator mask, and a medical universal mouthpiece. The sample collection subsystem may further include a containment booth for receiving an individual and isolating the individual's exhaled breath from ambient air, and the extraction component may be fluidly connected to the capture component through a wall of the containment booth. The diagnostic device may include at least one of PCR, rt-PCR, an immuno-based assay, a mass spectrometer (MS), MALDI-MS, ESI-MS, GC-MS, GC-IMS, and MALDI-TOFMS. The system may further include one or more cooling devices in thermal communication with the interface tube and a wall of at least one of the sample capture components and configured to cool the sample capture component. The sample capture component may be cooled to a temperature greater than about 0° C. and less than about 10° C. using the one or more cooling devices. The sample capture component can be cooled to a temperature greater than about 0° C. and less than about 4° C. using the one or more cooling devices. The one or more cooling devices can include a Peltier thermoelectric device. The system further includes one or more sensors configured in fluid communication with the sample extraction component, and an output of the one or more sensors can be used to calculate a total cumulative amount of exhaled aerosol particles entering the sample capture component. The one or more sensors can include a CO. 2 The diagnostic device may include at least one of a sensor, an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer. The number of exhalation maneuvers required to diagnose a respiratory disease using the diagnostic device may be determined using the total cumulative amount of exhaled aerosol particles.

[0017] The exemplary system may further include a sterilization component for sterilizing the sample collection subsystem. The sterilization component may include at least one of a nebulizer for spraying a sterilant, one or more UV lights for generating UV radiation, a steam generator, an ozone generator, a peroxide vapor generator, and combinations thereof. The sterilant may include at least one of 60% ethanol in water, at least 60% isopropanol in water, and a peroxide solution. The collected sample may be transferred to the sample processing component using at least one of a distribution pump, gravity induced flow, and a robotic sample transfer system. The pump may be a peristaltic pump. The interface tube may be fabricated from at least one of copper and nickel-copper alloy 400. The sample capture component may be fabricated from at least one of copper and nickel-copper alloy 400. The diagnostic device may include a MALDI-TOFMS. The sample processing component may include at least one of a fluid reservoir and a fluid distribution pump to distribute approximately one microliter of fluid onto the collected sample disposed on the sample substrate. The liquid may include at least one of a solvent, a MALDI matrix chemical, water, and an acid. The individual may be at least one of an individual infected with at least one of tuberculosis and coronavirus disease and an individual not infected. The lipid biomarkers may include biomarkers characteristic of Mtb. The sample capture element may include a packed bed column in fluid communication with the sample extraction component to selectively capture EBA particles. The packed bed column may include a packed bed column containing resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4 The packed bed column may include solid particles having at least one of the nanoparticles. The packed bed column may include resin beads having C18 functional groups on a surface thereof.

[0018] An autonomous method for diagnosing respiratory diseases in an individual using exhaled breath is disclosed, comprising the steps of: extracting EBA particles released from the individual during the predetermined number of breathing maneuvers into a flow of air that is fed to a sample extraction component configured to receive the face of the individual; collecting EBA particles from the exhaled breath and air as a collected sample using a sample capture component fluidly connected to the sample extraction component by an interface tube; spotting a small amount of the collected sample onto a sample plate; processing the sample by treating the sample with at least one of a solvent, MALDI matrix chemical, water, and acid, and mixtures thereof; and analyzing the sample using a diagnostic device. The EBA particles may include at least one of microbial, viral, metabolite biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of respiratory diseases. The processing step may further include concentrating the sample by drying the sample using a suitable drying means. The diagnostic device may include a MALDI-TOFMS. The step of collecting using the sample capture component may include flowing the output from the extracting step through a packed bed column to selectively capture EBA particles, and extracting the EBA particles from the packed bed column using at least one of about 12.5% ​​acetic acid, about 70% isopropanol, about 5% TFA, about 5% formic acid, and about 10% HCl in a sample extraction system to generate a collected sample. The method may further include digesting the collected sample to generate a peptide sample characteristic of EBA particles. The packed bed column may be composed of a resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4 The packed bed column may include solid particles having at least one of the nanoparticles. The packed bed column may include resin beads having C18 functional groups on a surface thereof.

[0019] An autonomous method for diagnosing respiratory diseases in an individual using exhaled breath is disclosed, comprising the steps of: instructing an individual to place a sample extraction component for extracting EBA particles from the exhaled breath; initiating a predetermined number of breath maneuvers to expel EBA particles from the exhaled breath into a stream of air that is fed to the sample extraction component; forcing EBA particles in the air into the sample capture component while cooling the walls of the sample capture component and an interface tube that fluidly connects the sample capture component to the sample capture component; generating a collected sample; processing the collected sample, including treating the sample with at least one of a solvent, a MALDI matrix chemical, water, and an acid, and mixtures thereof; and analyzing the plated sample using a MALDI-TOFMS. The flow rate of air entering the sample capture component can be between about 100 L / min and about 1000 L / min. The flow rate of air entering the sample capture component can be between about 50 L / min and about 500 L / min. The volume of the sample collected may be between about 100 microliters and about 1 ml. The predetermined number of breathing maneuvers may include taking a deep breath to clear the individual's lungs, pausing for up to 10 seconds, performing an FVC inhale, taking a deep breath, and repeating the sequence up to 10 times. The method may further include at least one of exhalation breathing, coughing, normal FVC breathing, speaking, and sneezing. The respiratory disease may include at least one of tuberculosis, influenza, pneumonia, cancer, and a disease caused by a coronavirus. The predetermined number of breathing maneuvers may be determined by one or more sensors indicative of at least one of a volume of exhaled particles and a volume of exhaled air. The one or more sensors may include CO 2 The sensor may include at least one of an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer.

[0020] An exemplary system for diagnosing respiratory diseases caused by aerosolized viral and bacterial particles is disclosed, the system comprising a sample capture component for collecting a predetermined volume of airborne EBM particles in an airflow provided to the sample capture component as a collected sample, the airflow being between about 30 L / min and about 1000 L / min, a sample analysis subsystem fluidly connected to the sample capture component, the sample analysis subsystem including a sample processing component for spotting a small amount of the collected sample onto a sample plate and handling the collected sample on the sample plate, and a diagnostic device for analyzing the sample. The sample processing component may include a fluidic component for processing the sample using at least one of a solvent, a MALDI matrix chemical, water, and an acid, and mixtures thereof. The system further comprises one or more sensors configured to be in fluid communication with the sample extraction component, and an output of the one or more sensors may be used to calculate a total cumulative amount of exhaled aerosol particles entering the sample capture component. The one or more sensors may include a CO 2 The predetermined volume of air can be determined using the output of one or more sensors.

[0021] Other features and advantages of the present disclosure are set forth in part in the following description and the accompanying drawings, in which preferred embodiments of the present disclosure are described and illustrated, and which will become apparent to those skilled in the art upon examination of the following detailed description in conjunction with the accompanying drawings, or may be learned through the practice of the present disclosure. The advantages of the present disclosure may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. [Brief description of the drawings]

[0022] The foregoing aspects and many of the attendant advantages of the present disclosure will be more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic diagram of an exemplary EBA-based diagnostic system. [Diagram 2] FIG. 2 is a schematic diagram of an exemplary EBA sample collection subsystem. [Diagram 3] FIG. 3 is a perspective view of a containment booth that may be optionally used with the EBA sample collection subsystem. [Figure 4A] Figure 4A is an ion chromatogram showing the differences in spectral signatures of volatile organic compounds in exhaled breath analysis between TB patients (Figure 4A) and non-TB patients (Figure 4B). [Figure 4B] Figure 4B is an ion chromatogram showing the difference in spectral signatures of volatile organic compounds in breath analysis between TB patients (Figure 4A) and non-TB patients (Figure 4B). [Diagram 5] FIG. 5 is a schematic diagram of an exemplary diagnostic method using exhaled breath aerosol (EBA) and exhaled breath condensate (EBC) analysis. [Figure 6A] FIG. 6A is one of the variations in particle size distribution in exhaled breath from three healthy individuals using a modified FVC breathing maneuver. [Figure 6B] FIG. 6B is one of the variations in particle size distribution in exhaled breath from three healthy individuals using a modified FVC breathing maneuver. [Figure 6C] FIG. 6C is one of the variations in particle size distribution in exhaled breath from three healthy individuals using a modified FVC breathing maneuver. [Figure 7] FIG. 7 shows exhaled carbon dioxide measurements during various breathing maneuvers. [Figure 8] FIG. 8 shows lung volumes collected from exhaled breaths during various breathing maneuvers. [Figure 9]Figure 9 shows the weighted principal component analysis (PCA) of the MS signals acquired from the positive and negative ion modes of the TB and non-TB samples.

[0023] All reference numbers, identifiers, and callouts in the figures are incorporated herein by this reference as if fully set forth herein. Failure to number elements in the figures is not intended as a waiver of any rights. Unnumbered references may also be identified by the alphabetical letter of the figure or appendix.

[0024] The following detailed description includes references to the accompanying drawings, which form a part of the detailed description. The drawings show, by way of illustration, specific embodiments in which the disclosed systems and methods may be practiced. These embodiments, which should be understood as "examples" or "options," are described in sufficient detail to enable one skilled in the art to practice the invention. The embodiments may be combined, other embodiments may be utilized, or structural or logical changes may be made without departing from the scope of the invention. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of the invention is defined by the appended claims and their legal equivalents.

[0025] In this disclosure, aerosol generally refers to a suspension of particles dispersed in air or gas. "Autonomous" diagnostic systems and methods mean that they generate diagnostic test results "without or minimal intervention by a medical professional." The U.S. FDA classifies medical devices based on the risk associated with them and by assessing the amount of regulation that would reasonably ensure their safety and effectiveness. Devices are classified into one of three regulatory classes: Class I, Class II, or Class III. Class I includes devices with the lowest risk, while Class III includes devices with the highest risk. All classes of devices are subject to general controls. General controls are a basic requirement of the Food, Drug, and Cosmetic (FD&C) Act that applies to all medical devices. In vitro diagnostic products are reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including the determination of health status, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and testing of specimens taken from the human body. The exemplary devices disclosed herein are capable of operating autonomously and producing reliable results, and as a result may be regulated as Class I devices. In some parts of the world where the burden of TB infection is high, access to medically trained personnel is very limited. Autonomous diagnostic systems are preferred over non-autonomous diagnostic systems.

[0026] The singular term (equivalent to the English terms "a" or "an") is used to include one or more, and the term "or" is used to refer to a non-exclusive "or" unless otherwise specified. Furthermore, it is to be understood that expressions or terms used herein and not otherwise defined are for descriptive purposes only and not for limiting purposes. Unless otherwise specified in this disclosure, for the purposes of interpreting the scope of the term "about", the error range associated with the disclosed values ​​(dimensions, operating conditions, etc.) is ±10% of the value stated in this disclosure. The error range associated with values ​​disclosed as percentages is ±1% of the stated percentage. The word "substantially" used before certain words includes the meanings "a significant portion of the specified range" and "most but not all of what is specified". DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Particular aspects of the invention are described in some detail below for purposes of explaining the composition, principles, and operation of the disclosed methods and systems, however, various modifications may be made and the scope of the invention is not limited to the exemplary aspects described.

[0028] An exemplary exhaled breath analysis ("EBA") based diagnostic system 100 can include an EBA sample collection subsystem 101, and an analysis subsystem 102 (FIGS. 1-3). These two subsystems are described in more detail below.

[0029] [EBA Sample Collection Subsystem 101] The subsystem 101 may include a sample extraction component 104, which may be in the form of at least one of a shroud and a loose-fitting cone-shaped device. A suitable form-fitting mask adapted to receive the face of the individual or removably attachable to the face / head of the patient / individual 105 using straps or the like may also be used, but is not preferred due to the difficulty in ensuring a good fit for all humans, especially men with beards / facial hair. The individual may be seated in an optional containment booth 106 to isolate the patient's EBA from the ambient air in the testing room or area. An exemplary containment booth 106 may include a modified respiratory function testing body box, such as that sold by Morgan Scientific Inc. (Haverhill 9, Massachusetts), by replacing the plethysmograph component with the extraction component 104 described herein, such that the booth 106 is in fluid communication with the extraction component 104. The booth 106 may also be a modified version of the Respiratory Aerosol Sampling Chamber (RASC) chamber described by Wood et al. (2016) and incorporate the features and capabilities described therein. Wood et al. (2016) is a non-patent document cited in the References section, the disclosure of which is incorporated herein by reference in its entirety. In the RASC chamber, participants are seated and passively engaged in a breath sampling protocol. Following the steps outlined in Wood et al., approximately one hour is spent within the chamber. Briefly, the chamber is sealed and an air purge phase is performed in which ambient air is entrained through a high-efficiency particulate arrestance (HEPA) filter for 10 minutes. This is followed by a participant-led contamination phase in which the chamber is isolated from the outside environment and a proportion of the exhaled air is purged with CO2 from the chamber at 4,000 ppm above ambient levels. 2The concentration is allowed to rise to a threshold of 10% defined by the assumed exhaled CO 2 (When the concentration is 40,000 ppm) Measured CO 2can be used to calculate the exhaled volume, as described by Wood et al. If the target is not reached after 30 min, begin the sampling phase with a lower exhalation rate. After sampling, the chamber is purged again to remove residual Mtb from the air. Contamination of the sampling chamber was primarily caused by periodic breathing, in addition to spontaneous coughing and sneezing. Particles and organisms originating from sources other than breathing were minimized by participants wearing full-body DuPont Tyvek suits during sampling, and an initial purging phase to minimize ambient contamination. Component 104 serves to extract aerosol particles expelled from the mouth and nose of the patient 105 into an air stream that acts as a sheath fluid (usually air) supplied by the air source 107, helping to transport the aerosol towards the outlet of component 104 and introduce it into the sample capture component 108 without deposition on the walls of component 104. Air source 107 can be an air pump or compressor. Examples of component 104 include the funnel-shaped cone used by Milton's group and the face mask used by Fennelly. The air sheath fluid can be added through the walls of the component 104 or at the edges, or more commonly to the booth 106. The airflow supplied to the component 104 can be appropriately filtered (e.g., using a HEPA filter) to remove all or nearly all particulate matter, including but not limited to dust and infectious agents, in the ambient air. HEPA stands for High Efficiency Particulate Air, a designation used to describe a filter capable of trapping 99.97% of particles 0.3 microns in size, and is used to remove all or nearly all particulate matter in the ambient air, including but not limited to dust and soot. Additionally, the sheath fluid flow can be humidified to allow for EBA particle size growth, thereby allowing a majority of the exhaled particles to be captured downstream of the aerosol capture device 108.An interface tube 109 fluidly connects the extraction component 104 to the sample capture component 108 and allows for further cooling to grow the size of the EBA particles. Cooling may be provided using a refrigeration system or, more preferably, using a Peltier thermoelectric cooler 113, which is small, lightweight, and consumes little power to operate. For example, Peltier coolers supplied by Marlow Industries (Dallas, TX), TE Technologies Inc. (Traverse City, MI) typically include an array of alternating n-type and p-type semiconductors. The array is soldered between two ceramic plates, electrically in series and thermally in parallel. Bismuth telluride, antimony telluride, and bismuth selenide are suitable materials for Peltier effect devices, as they offer the best performance from 180K to 400K and can be made into both n-type and p-type. The Peltier effect creates a temperature difference by transferring heat between two electrical junctions when a voltage is applied to the coupled alternating n-type and p-type semiconductors to create an electric current. Heat is removed at one junction, providing cooling; heat is stored at the other junction, where it can be easily removed with a fan or blower.

[0030] The components 104 may be disposable to limit the risk of a patient becoming contaminated or infected with pathogens released by a previous patient. Alternatively, the components 104 may be reusable, in which case they may be sterilized using a sterilization component 110 using at least one of a disinfectant spray rinse produced using a suitable nebulizer, UV radiation, a peroxide solution or steam treatment, steam sterilization, or a combination thereof. A nebulizer, for example an impingement type nebulizer (supplied by CH Technologies), may be fluidly connected near the exhaust of the cone, i.e., near the throat region 111. A cleaning fluid is sprayed to disinfect the extraction component 104, the tube 109, and the capture component 108. The cleaning fluid is selected to ensure that the EBA and condensed EBA (exhaled breath condensate) sample and the components within the sample collection subsystem 101 remain generally sterile. For example, if 70% ethanol in isopropyl alcohol is used, this disinfectant solution can be easily removed from the sample by evaporation and does not interfere with the analysis. The sterilization component 110 (e.g., nebulizer) can be briefly activated at the end of the sample collection period to provide a final wash of the inlet tube 109 and capture component 108. The nebulizer 110 can be activated again to wash the sample extraction component 108 before reuse with the next patient. Waste liquid can be pumped using pump 116 to an acceptable waste by switching valve 117 to fluidly connect pump 116 to a waste container. Although the exemplary sample extraction component 104 and sample capture component 108 can be disinfected between patients, the exemplary system 100 with MS diagnostic device does not require 100% decontamination of the exemplary system between different individuals due to its high sensitivity to bioaerosol fragments of interest even in the presence of trace contaminants.

[0031] Although the extraction component 104 and tube 109 are shown as converging / diverging in diameter, the diameter of the tube may be the same as the diameter of the throat 111 of the component 104, or may be larger or smaller than the diameter of the throat 111. The sensor 112 may be, but is not limited to, a CO 2 The sensor 112 may be fluidly connected to a component 104 near the throat region 111, which includes a sensor and a particle sizer / counter. The sensor 112 provides an indication of the volume of exhaled air sampled. 2 Monitoring allows for a close approximation of the percentage of exhaled volume for each participant within the containment booth 106 at any given time. For example, if lung capacity declines and forced vital capacity (FVC) is relatively small (e.g., less than 2 liters) or coughs are weak (e.g., less than 1 liter of exhalation), the patient is automatically and in real time instructed to provide more FVC breaths or coughs until a sufficient amount of exhaled aerosol is collected. Wurie (2016) describes bioaerosol production by tuberculosis patients during normal breathing and its impact on infection risk. Optical particle counter technology was used to measure aerosol size and concentration over time during 15 exhalations (ranging from 0.3 to 20 μm in diameter) in four groups (healthy / non-infected, healthy IMtb-infected, patients with extrathoracic TB, and patients with intrathoracic TB). High particle generation was defined as bioaerosol counts of 1 to 5 μm size above the median for all participants (median count = 2 counts / L). Data from 188 participants were obtained before treatment (baseline). Bioaerosol production varied considerably between individuals. Multivariate analysis showed that intrathoracic tuberculosis was associated with a 3.5-fold increased odds of high production of 1-5 mm bioaerosols compared to healthy / uninfected individuals. Wurie (2016), a non-patent publication cited in the References section, is incorporated herein by reference in its entirety.

[0032] The EBA sample capture component 108 may be a wet wall cyclone (as shown in FIG. 2), one or more impactors (e.g., as shown by Milton), or impingers, which use the following dry or near-dry collection methods: Washing resuspends EBA particles from the collection surface of the cyclone. Exemplary capture components 108 include, but are not limited to, wet film impactors (McDevitt, 2013), Coriolis™ wet wall cyclones (Bertin, France), impingers such as BioSampanr (SKC, Inc, Eight-Four, PA), impaction-based devices such as BioCapture (FLIR Systems, OR), 300 L / min wet wall cyclones (King, 2012), and BioSpot Sampler (Aerosol Devices, Fort Collins, CO). McDevitt's wet film impactor and BioSpotSampler use a combination of humidification and condensation to "grow" the size of aerosol particles, thereby increasing the collection efficiency of submicron-sized particles in EBA. As cited in the References section, each of the non-patent publications by McDevitt and King and the contents therein are incorporated herein by reference in their entirety. EBA aerosol particles can be collected directly in a liquid, or collected on a filter media and extracted by backflushing water or solvent through the filter, dissolving the filter using a dissolvable filter material, or grinding the filter into a liquid, after which the resulting slurry can be analyzed. EBA aerosol particles can be collected by impacting the particles against a dry surface and then washing the particles from the surface with an appropriate fluid. Virtual impactors can be used to concentrate aerosols of a particular "cut size" and aerosols larger than the cut size. Virtual impactors, such as those described in U.S. Pat. No. 6,062,392, can be combined with impactors and other devices, such as those shown in U.S. Pat. No. 6,267,016, to increase the inlet airflow rate of air, including exhaled air.The sample capture component 108 may include a condensation growth tube for growing sub-micron particles into micron-sized particles. Biomarkers may include lipids from Mtb cell walls, and these lipids, in addition to Mtb cells, may be used for disease diagnosis. Nearly 100% of the breath sample is collected. There is no need to dilute the collected sample 115 with saline.

[0033] The EBA aerosol particle capture component 108 can have a flow rate of sheath fluid (air) between about 100 L / min and about 1000 L / min. The air flow rate is preferably greater than 200 L / min and is about 300 L / min. McDevitt used a flow rate of 130 L / min, which is insufficient to reliably capture EBA generated during coughing. A high flow rate minimizes aerosol loss due to blowback during the coughing maneuver. A higher flow rate leads to more entrainment of EBA particles. Preferably, the aerosol capture component 108 collects particles into a small condensed sample 115, thus producing a concentrated aerosol biomass (e.g., at least 1 nanoliter of peripheral lung fluid per milliliter of collected fluid). The sample 115 can be transferred to the analysis subsystem 102 using a pump 116, which is preferably a peristaltic pump. The valve 116 can be used to send the condensed sample 115 to the analysis subsystem 102 or to a waste container, for example, during decontamination of the sample collection subsystem 101. A volume of EBA sample liquid of less than about 1 ml is preferred and targeted. The exemplary disclosed system may be capable of producing between about 100 microliters and about 200 microliters of fluid. Thus, not all of the exemplary EBA sample capture components identified herein are preferred for use in the disclosed exemplary sample collection subsystem 101 for an autonomous system. For example, the BioSampler and Coriolis aerosol samplers collect EBA aerosol particles in aqueous samples with volumes of over 10 ml. This large volume results in a very dilute sample, necessitating particle concentration methods. A preferred aerosol capture component 108 would have a high inlet air flow rate to entrain a large proportion of particles in the exhaled breath, even during coughing, when the exhaled sneeze flow rate is very non-uniform in time. Similarly, the McDevitf wet film impactor injects steam upstream of the impactor and condenses it to provide the sample. The sample is collected in a 50 ml centrifuge tube and concentrated using a centrifuge. As previously mentioned, the exemplary EBA sample collection subsystem captures particles in a liquid volume of about 1 ml or less.The component 108 is preferably cooled using one or more Peltier cooling devices 114, as is the cooling tube 109 that fluidly connects the extraction component 104 to the component 108 using a Peltier device, allowing the EBA particles in the exhaled breath to grow in size articles formed deep in the lungs. Although diameters can be on the order of 100 microns, they can grow to over 1 micron. The cooling of the component 108 and tube 109 promotes condensation of volatile compounds in the exhaled breath that are also collected in the liquid sample. Thus, the exemplary EBA capture component 108 collects both volatile and non-volatile biomass in the exhaled breath. If a cyclone is used as the capture component 108, the cyclone and cyclone inlet tube are preferably manufactured from copper, a copper alloy such as nickel-copper alloy 400, or other alloys with high thermal conductivity and low cost. Additionally, copper and copper alloys have inherent antibacterial properties. Peltier coolers 113 and 114 are preferred as cooling devices because it is easy and precise to control the temperature of the cyclone inlet 109 and the body of the cyclone 108. Entrained air supplied to the sample capture component 108 (e.g., when 108 is a cyclone) may be supplied using pump 118 and filtered using HEPA filter 119.

[0034] [EBA Sample Analysis Subsystem 102] The EBA liquid sample 115 containing the EBA aerosol particles is then sent to a sample processing component 120 for analysis using at least one of a diagnostic device 112 for analyzing EBA particles and a device 122 for analyzing volatile organics in the exhaled breath. The sample processing component 120 may include the elements necessary to perform one or more of the following steps:

[0035] (a) Sample 115 may be placed in a cup or vial, for example, the Series 110A Spot Sampler (Aerosol Devices) uses a 32-well plate with either a circular well geometry (75 μL well volume) or a teardrop well geometry (120 μL well volume) and is heated to evaporate excess liquid / fluid within the sample, concentrating the sample.

[0036] (b) The sample 115 may be placed in a cup and exposed to a vacuum source to evaporate the fluid and concentrate the sample.

[0037] (c) The sample 115 may be mixed with a highly volatile solvent (eg, methanol, ethanol, and acetonitrile) to speed up the evaporation process.

[0038] (d) The sample 115 can be subjected to bead-based extraction. Bead-based extraction can be used to extract biomarkers from dilute solutions. For example, micron-sized magnetic beads can be coated with a glycan material that binds well to protein biomarkers, such as EBA particles. The beads can be intimately mixed with the EBA sample by an oscillating magnetic field. After mixing for a period of time, the beads can be pulled to one side by a constant magnetic field and then released into a small volume of solvent to extract the EBA particles as a concentrated sample.

[0039] (e) The sample 115 may be subjected to a solvent extraction process in which the sample is in intimate contact with an immiscible fluid such that the biomarkers (EBA particles) are preferentially transferred to the immiscible fluid. For example, a relatively large aqueous collection fluid sample (>1 ml) may be contacted with a relatively small amount of organic solvent (e.g., hexane or chloroform), which transfers the EBA particles and cell fragments in the sample from the matrix to the organic phase.

[0040] Many diagnostic devices may be adapted for use in the analytical subsystem 102, including, but not limited to, devices that perform genomics-based assays (such as PCR, rt-PCR, and whole genome sequencing), biomarker recognition assays (such as ELISA), and spectral analysis such as mass spectrometry (MS). Of these diagnostic instruments, MS is preferred for its speed of analysis. Suitable MS techniques for biomarker identification are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) MS. ESI can be combined with high-resolution mass spectrometers such as Oribtrap (ThermoFisher). ESI-MS devices are typically very large and heavy, require a high level of expertise to operate, and are not suitable for applications such as autonomous operation or point-of-care diagnostics. In contrast, MALDI-MS devices are compact and lightweight, consume less than 100 watts of power, and provide sample analysis in less than 15 minutes. MALDI-MS is a suitable diagnostic device for point-of-care diagnostics suitable for ACF. Including the time for sample preparation, the analysis time using MS can be less than about 15 minutes. Samples must be dried before insertion into the MALDI spectrometer, and large (>1 ml) samples cannot be dried quickly without loss or decomposition of the analytes. For concentrated samples,115 sample analysis using MALDI MS can take less than 5 minutes (including sample preparation) due to the short time required to evaporate water from the sample.

[0041] "Matrix Assisted Laser Desorption Ionization" (MALDI) allows large molecules to be analyzed intact using mass spectrometry. In this technique, the target particle (analyte) is coated with a matrix chemical. The matrix chemical preferentially absorbs light from a laser (often ultraviolet wavelengths). In the absence of a matrix, biomolecules break down by pyrolysis when exposed to a laser beam in a mass spectrometer. The matrix chemical also transfers charge to the vaporized molecules, producing ions. The ions are accelerated down a flight tube by an electric field. Microbiology and proteomics have become major application areas for mass spectrometry. Examples include bacterial identification, discovery of chemical structure, and derivation of protein function. MALDI-MS has also been used for lipid profiling of algae. In MALDI-MS, a liquid consisting of an acid such as trifluoroacetic acid (TFA) and a MALDI matrix chemical such as α-cyano-4-hydroxycinnamic acid is added to the sample in a solvent, usually dissolved in water. Solvents include acetonitrile, water, ethanol, and acetone. TFA is usually added to suppress the effect of salt impurities on the mass spectrum of the sample. Water allows for the dissolution of hydrophilic proteins, while acetonitrile allows for the dissolution of hydrophobic proteins. The MALDI matrix solution is spotted onto the sample on the MALDI plate to produce a uniform, homogenous layer of MALDI matrix material on the sample. The solvent evaporates, leaving only the recrystallized matrix, and the sample spreads throughout the matrix crystals. The acid partially degrades the cell membrane of the sample, making the proteins available for ionization and analysis in the MS. Other MALDI matrix materials include 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), α-cyano-4-hydroxycinnamic acid (α-cyano or α-matrix), and 2,5-dihydroxybenzoic acid (DHB), which are described in U.S. Patent No. 8,409,870.

[0042] In the exemplary system 100, the use of a chiller to cool the capture component 108 and the inlet line 109 produces a sample 115, which includes a cooled liquid sample containing condensed volatile organic compounds and EBA in the exhaled breath. Thus, the sample 115 can be sent to a diagnostic device 122 for analysis of condensed volatile organic compounds and to the device 112 for analysis of non-volatile EBA particles. During sample processing in step 204 (FIG. 5), the liquid sample 115 can be warmed using a heater to provide the volatile compounds to a diagnostic device 122, for example, GC-MS, GC-IMS, volatile ion chromatography, or any other analytical method suitable for analysis of volatile organic compounds. FIG. 4 shows that ion chromatograms from exhaled breath can be used to distinguish healthy individuals from tuberculosis-infected patients. Hashoul (2019) discloses the use of sensors to detect pulmonary diseases, including tuberculosis, from exhaled breath. Hashoul describes an analysis of exhaled breath of 226 symptomatic high-risk patients using GC (gas chromatography)-MS and points out several biomarkers of active pulmonary tuberculosis. They proposed biomarkers of oxidative stress products such as alkanes and alkane derivatives, and volatile metabolites of M. tuberculosis such as cyclohexane and benzene derivatives. Their results distinguished between positive and negative TB with an overall accuracy of 85%, sensitivity of 84%, and specificity of 64.7% using the C-statistic. Metal oxide sensors were used. Furthermore, the sensor array had a sensitivity of 93.5% and specificity of 85.3% in distinguishing between healthy controls and TB patients, and a sensitivity of 76.5% and specificity of 87.2% in identifying TB patients across the entire test population. The use of gold nanoparticles (GNPs) and QMB (quartz crystal microbalance sensors) was also discussed. The non-patent literature published by Hashoul cited in the References section is incorporated herein in its entirety. If cooling is not used in subsystem 101 as previously mentioned, the liquid sample 115 may not contain condensed volatile organic compounds. As a result, the diagnostic device may be limited to analyzing EBA particles in liquids using device 121.

[0043] FIG. 5 is a schematic diagram of an exemplary diagnostic method 200 using the exemplary system 100 previously disclosed herein. Using the exemplary method 200, an autonomous point-of-care diagnosis based on exhaled breath can be performed. In step 201, an individual 105 can be instructed to sit. The chair can be optionally placed in a containment booth 106. In step 202, an extraction component 104 can be removably attached to the individual's head, or a cone larger than the head can be placed to fit loosely around the individual's head. The sample capture component 108 is activated, which draws air around the patient's head and into the sample capture device. If a cyclone is used as the sample capture component 108, air flow to the component 108 and cooling of the cyclone body and inlet tube 109 are initiated. Preferably, filtered sheath air is provided to the component 104. The sheath air can be humidified, preferably to a relative humidity of more than 90%. The individual 105 is then instructed to perform one or more of the above-described predetermined operations 203, which may include a preset number of repetitions. The operations include performing one or more FVC or modified FVC operations to generate an EBA sample from the lower airways, generating one or more cough samples to generate an EBA primarily from the upper airways, and generating one or more sneeze samples to generate an EBA primarily from the nasal / upper airways. A modified FVC is an FVC preceded by a deep exhalation followed by a 5-10 second pause. This exhalation and pause causes the bronchioles to close and then open again during FVC inhalation. The closing and reopening of the small lung passages, including the alveoli, is believed to result in increased particle production. For TB diagnosis using the exemplary system 100, about 3 to about 5 repetitions of the modified FVC may be required. The operations may include coughing, FVC breathing, modified FVC breathing, and sneezing. For diagnosis of some other diseases, all operations may be required. Sneezing may be induced by injecting small amounts of pepper or other spices in aerosol form into the nasal passages.Preferably, at least three modified FVC breaths to sample the lower airway and a series of coughs to sample the upper airway may be required. The lower airway generally consists of the trachea, lungs, and all parts of the bronchial tree (including the alveoli), with the organs of the lower airway located within the thoracic cavity. The upper airway generally consists of the nose, pharynx, and larynx, with the organs of the upper airway located outside the thoracic cavity. In some cases, at least 10 modified FVC breaths may be required. To sample the nasal cavity, a series of sneezes may be induced. A preferred modified FVC that increases biomass collection per breath is one in which the patient first exhales, then pauses for up to 10 seconds before an FVC inhalation, followed by a full exhalation as disclosed by Bake et al. (2019), a non-patent publication cited in the References section and incorporated herein by reference in its entirety. A preferred breathing maneuver may include a 10x cough / FVC / bronchial blast. Instead of a cone or shroud, the sampling component 104 may include a continuous positive airway pressure (CPAP) mask used to treat sleep apnea (e.g., the Dreamware and Amara masks sold by Philips Respironics). CPAP works by blowing air into the throat through a mask, subtly increasing air pressure in the throat and preventing the airway from narrowing, but has been modified to collect exhaled air from various breathing maneuvers, using the airflow as a sheath fluid.

[0044] The patient is then instructed to leave the chair and take a seat in the waiting area. The extraction component 104 can be disinfected with UV light. In a sample processing step 204, the sample 115 is automatically transferred from the collection subsystem 101 for sample processing in the analysis subsystem 102. The type of sample processing depends on the type of diagnostic device. If the diagnostic device is a MALDI-MS, the sample processing can include plating the sample 115 onto a MALDI-MS sample disk using a peristaltic pump, heating the disk to concentrate the sample, adding MALDI matrix / acid / solvent (described below), and drying the disk. The sample disk is then analyzed using MALDI-MS in step 205. The resulting spectrum is compared to spectra of samples known to be positive for a particular respiratory infection and to spectra of samples from patients known to be healthy, and a diagnosis for the patient is formed. The results can then be communicated to the clinician or patient. Once the extraction component 204 is attached to the patient and sample extraction is initiated, the exemplary method is autonomous after performing the necessary operations (except for asking the patient to leave the chair) and produces a diagnostic test result.

[0045] With regard to the FVC maneuver in step 203, Figures 6A-C show the normal variability of exhaled aerosol from a healthy individual for 10 repetitions of the FVC breathing maneuver. Even in healthy individuals, there is a great deal of variability in the amount and particle size distribution of exhaled aerosol. Data were captured using a LASEX II (PMS, City, CO). Similar variability was seen during EBA collection from the cough maneuver. The particle size distribution helps determine the total exhaled particle mass by integrating the particle size distribution over time. This aspect helps determine whether the collected sample is sufficient for analysis. Figure 7 shows the carbon dioxide measurements in the exhaled air during the bronchiolar film burst (BB), FCV and cough maneuver. According to Patterson et al. 2A strong correlation was observed between the production rate and aerosol particle production. As described in Wood et al., the measured CO 2 can also be used to calculate exhaled volume.

[0046] Figure 8 shows the amount of exhaled fluid volume (in nanoliters) based on particle size distribution and particle concentration, as measured by the LASEX device. The modified FVC maneuver is preferred over the standard FVC, as it is the method that provides the largest sample, as the preferred method. Coughing provides a similar amount of EBA sample volume, primarily from the upper respiratory tract. Both of these methods generate sufficient samples to detect tuberculosis infection in patients known to be infected but not yet receiving treatment.

[0047] Regarding the use of MS to detect biomarkers of TB infection in exhaled breath, positive and negative ion signals containing thousands of features were acquired (or extracted) using a high-resolution Orbitrap mass spectrometer (ThermoFisher Scientific) from samples collected from TB patients (n=20), and non-TB / control samples (n=13). Masses with a signal-to-noise ratio (SNR) greater than 5:1 were selected. A large set of signals was reduced to two components using weighted principal component analysis (PCA), an unsupervised dimensionality reduction algorithm. PCA provides a 2D visualization, which was used to investigate whether the extracted signals reveal intrinsic differences between the two classes of samples (TB and non-TB). The PCA results (Figure 9) revealed that samples from each group tended to cluster together, suggesting that the extracted signals collected from high-resolution mass spectrometry can be used to distinguish between the two classes of samples.

[0048] The exemplary systems and methods described herein are not necessarily limited in their diagnostic capabilities to respiratory infections. For example, lung cancer may also release biomarkers into peripheral lung fluid, and these biomarkers would be easily detected by the disclosed systems and methods. Furthermore, because blood is in close contact with the alveolar lining of the lungs, biomarkers of infections and cancers in other parts of the body (beyond the lungs) may migrate beyond the alveolar lining into peripheral lung fluid and would be detected by analysis of EBA. As a result, the scope of the present invention is not limited to the detection and diagnosis of respiratory diseases.

[0049] An exemplary sample extraction component may include a packed bed column for selectively capturing non-volatile organic components, including EBA particles, in exhaled breath. The non-volatile components in exhaled breath may include exhaled aerosol particles, including at least one of microorganisms, viruses, metabolic biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of respiratory disease. The packed bed column may be made of a resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4The packed bed column may include solid particles including at least one of nanoparticles. The packed bed column may include resin beads having octadecyl acrylate (C18) functional groups on a surface. The resin beads may have a nominal diameter between about 12 μm and about 20 μm. The solid particles may include functional groups immobilized on a surface of the particles, where the functional groups include at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethyl-aminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, an ion exchange phase, a polymer phase, an antibody, a glycan, a lipid, DNA, and RNA. The ion exchange phase may include at least one of diethylaminoethyl cellulose, QAE Sephadex, Q Sepharose, and carboxymethyl cellulose. The polymer phase includes at least one of polystyrene-co-1,4-divinylbenzene, methacrylate, polyvinyl alcohol, starch, and agarose. The antibodies may include at least one of anti-human albumin, anti-influenza A virus NP, and anti-SARS-CoV-2 virus. The antibodies may be immobilized on protein A / G agarose beads. The capture element may be cooled to a temperature below ambient temperature. Applicant's U.S. Provisional Patent Application No. 63 / 069,029, entitled "Diagnosis of Respiratory Disease Using Exhaled Breath and Aerosol Analysis," provides additional details related to exemplary sample extraction components including packed bed columns, and is incorporated herein by reference in its entirety.

[0050] Exemplary systems and methods disclosed herein may include robotic systems and components. For example, the systems and methods may include a robotic sample transport system for spotting collected samples onto a sample plate and performing further processing or sample handling and analysis of the processed samples. Commonly owned U.S. Provisional Patent Application No. 62 / 931,200, entitled "System and Method for Rapid and Autonomous Detection of Aerosol Particles," provides additional details related to exemplary robotic sample transport systems and is incorporated herein by reference in its entirety.

[0051] The collected sample does not need to be spotted on a sample plate prior to processing and analysis. For example, EBA particles in a collected liquid sample can be aerosolized using a nebulizer and coated "on the fly" using a MALDI matrix to form coated aerosol EBA particles. The coated particles can be analyzed using aerosol time-of-flight mass spectrometry (ATOFMS). "On the fly" means that the particles that make up the aerosol are not collected on a surface (e.g., the surface of a MALDI plate) or in a liquid as a step in the coating process. Applicant's U.S. Patent Application No. 15 / 755,063, entitled "Coating Aerosol Particles Using Acoustic Coating," provides additional details and is incorporated herein by reference in its entirety.

[0052] The Abstract is provided to comply with 37 C.FR § 1.72(b) to enable the reader to quickly determine the nature and gist of the technical disclosure from a general overview perspective, and is not intended to be used to interpret or limit the scope or meaning of the claims.

[0053] Although the present disclosure has been described in connection with preferred modes of carrying out the same, those skilled in the art will appreciate that numerous modifications can be made thereto without departing from the spirit of the disclosure, and therefore, it is not intended that the scope of the present disclosure be limited by the foregoing description.

[0054] It is also understood that various modifications can be made without departing from the essence of the present disclosure. Such modifications are implicitly included in the description. They still fall within the scope of this disclosure. It is understood that this disclosure is intended to yield patents covering many aspects of the disclosure, both independently and as a system as a whole, and in method and apparatus modes.

[0055] Moreover, each of the various elements of the disclosure and claims may also be achieved in a variety of ways, and the disclosure should be understood to encompass each such variation, whether that variation be in any apparatus implementation, method or process implementation, or simply variations of any of these elements.

[0056] In particular, it should be understood that each element word may be expressed by equivalent apparatus terms or method terms even if only the function or result is the same. Such equivalent, broader, or more general terms should be considered included in the description of each element or operation. Such terms may be substituted where necessary to make clear the implicitly broader scope to which this disclosure is entitled. It should be understood that every operation may be expressed as a means for taking that operation or as an element that causes that operation. Similarly, each physical element disclosed should be understood to encompass a disclosure of the operation that the physical element facilitates.

[0057] Furthermore, for each term used, unless its usage in this application is inconsistent with such interpretation, a common dictionary definition, as contained in, for example, at least one of the standard technical dictionaries recognized by artisans and the most recent edition of Random House Webster's Unabridged Dictionary, should be understood to be incorporated herein for each term and all definitions, alternative terms, and synonyms.

[0058] Additionally, the use of the transitional phrase "comprising" is used to maintain the "open-ended" claims herein in accordance with conventional claim interpretation. Thus, unless the context requires otherwise, "comprising" is intended to mean the inclusion of a recited element or step or group of elements or steps, but not the exclusion of other elements or steps or group of elements or steps. Such terms should be interpreted in the broadest manner to afford the applicant the broadest scope legally permissible.

[0059] The technical features described herein are listed below. [Technical feature 1] 1. An autonomous system for diagnosing a respiratory disease in an individual using exhaled breath, comprising: The system is a sample collection subsystem, a sampling component configured to receive the face of the individual to sample exhaled breath aerosol (EBA) particles emitted from the individual during the predetermined number of breathing maneuvers into the airflow provided to the sampling component; a sample collection subsystem having a sample capture component fluidly connected to the sample extraction component by an interface tube and configured to separate and collect EBA particles from the exhaled breath and air as a collected sample; a sample analysis subsystem fluidly connected to the sample capture component, a sample processing component for spotting a small amount of the collected sample onto a sample plate and concentrating the collected sample onto the sample plate; and a sample analysis subsystem having a diagnostic device for analyzing the sample. [Technical feature 2] The system described in technical feature 1, wherein the EBA particles have at least one of a microbial, viral, metabolic, lipid, and proteomic biomarker characteristic of the respiratory disease. [Technical feature 3] The system according to technical feature 1, wherein the flow rate of air entering the sample capture component is between about 100 L / min and about 1000 L / min. [Technical feature 4] The system according to technical feature 1, wherein the flow rate of air entering the sample capture component is between about 50 L / min and about 500 L / min. [Technical feature 5] The system of technical feature 1, wherein the collected sample volume is between about 100 microliters and about 1 ml. [Technical feature 6] The system according to technical feature 1, wherein the sample capture component further comprises an air pump and an impactor, the air pump providing an air flow to transport the exhaled breath from the extraction component to the impactor, and the impactor separating the EBA particles from the exhaled breath to generate a collected sample. [Technical feature 7] 7. The system according to technical feature 6, wherein the impactor comprises at least one of a cyclone, a wetted wall cyclone, one or more wetted film impactors, and an impinger. [Technical feature 8] 7. The system according to claim 6, further comprising at least one virtual impaction stage disposed upstream of the impactor. [Technical feature 9] The system according to technical feature 1, wherein the sample extraction component has at least one of a cone-shaped device, a shroud, a CPR rescue mask, a CPAP mask, a ventilator mask, and a medical universal mouthpiece. [Technical feature 10] The system of technical feature 1, wherein the sample collection subsystem further includes a containment booth for receiving an individual and isolating the individual's exhaled breath from ambient air, and the extraction component is fluidly connected to the capture component through a wall of the containment booth. [Technical feature 11] The system according to technical feature 1, wherein the diagnostic device comprises at least one of PCR, rt-PCR, an immuno-based assay, a mass spectrometer (MS), MALDI-MS, ESI-MS, GC-MS, GC-IMS and MALDI-TOFMS. [Technical feature 12] The system according to technical feature 1, further comprising one or more cooling devices in thermal communication with the interface tube and at least one wall of the sample capture component and configured to cool the sample capture component. [Technical feature 13] 13. The system according to technical feature 12, wherein the sample capture component is cooled to a temperature greater than about 0° C. and less than about 10° C. using the one or more cooling devices. [Technical feature 14] 13. The system according to technical feature 12, wherein the sample capture component is cooled to a temperature greater than about 0° C. and less than about 4° C. using the one or more cooling devices. [Technical feature 15] 13. The system according to claim 12, wherein the one or more cooling devices include a Peltier thermoelectric device. [Technical feature 16] The system described in technical feature 1 further comprises one or more sensors configured in fluid communication with the sample extraction component, the output of which is used to calculate the total cumulative amount of exhaled aerosol particles entering the sample capture component. [Technical feature 17] The sensor or sensors 2The system according to technical feature 16, having at least one of a sensor, an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer. [Technical feature 18] The system according to technical feature 16, wherein the number of exhalation maneuvers required to diagnose a respiratory disease using the diagnostic device is determined using the total cumulative amount of exhaled aerosol particles. [Technical feature 19] The system according to technical feature 1, further comprising a sterilization component for disinfecting the sample collection subsystem. [Technical feature 20] The system according to technical feature 19, wherein the sterilization component has at least one of a nebulizer for spraying a disinfectant, one or more UV lights for generating UV radiation, a steam generator, an ozone generator, a peroxide steam generator, and combinations thereof. [Technical feature 21] The system according to technical feature 20, wherein the disinfectant comprises at least one of 60% ethanol in water, at least 60% isopropanol in water, and a peroxide solution. [Technical feature 22] The system according to technical feature 1, wherein the collected sample is transferred to the sample processing component using at least one of a distribution pump, a gravity-induced flow, and a robotic sample transfer system. [Technical feature 23] 23. The system according to technical feature 22, wherein the pump is a peristaltic pump. [Technical feature 24] The system according to technical feature 1, wherein the interface tube is made of at least one of copper and nickel-copper alloy 400. [Technical feature 25] The system according to technical feature 1, wherein the sample capture component is made of at least one of copper and nickel-copper alloy 400. [Technical feature 26] The system according to technical feature 1, wherein the diagnostic device comprises a MALDI-TOFMS. [Technical feature 27] The system of technical feature 1, wherein the sample processing component has at least one of a fluid reservoir and a fluid distribution pump and distributes approximately 1 microliter of fluid onto the collected sample disposed on the sample substrate. [Technical feature 28] 28. The system according to technical feature 27, wherein the liquid comprises at least one of a solvent, a MALDI matrix chemical, water, and an acid. [Technical feature 29] The system according to technical feature 1, wherein the individual is at least one of a person infected with at least one of tuberculosis and coronavirus diseases, and a person who is not infected. [Technical feature 30] The system according to technical feature 2, wherein the lipid biomarker has a biomarker characteristic of Mtb. [Technical feature 31] The system according to technical feature 1, wherein the sample capture element has a packed bed column in fluid communication with the sample extraction component and selectively captures EBA particles. [Technical feature 32] The packed bed column is made of resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4 32. The system according to technical feature 31, having solid particles having at least one of the nanoparticles. [Technical feature 33] 32. The system according to technical feature 31, wherein the packed bed column has resin beads having C18 functional groups on the surface. [Technical feature 34] 1. An autonomous method for diagnosing a respiratory disease in an individual using exhaled breath, comprising: The method comprises: extracting EBA particles expelled from the individual during said predetermined number of breathing maneuvers into a stream of air that is delivered to a sampling component configured to receive the individual's face; collecting EBA particles from the exhaled breath and air as a collected sample using a sample capture component fluidly connected to said sample extraction component by an interface tube; spotting a small amount of the collected sample onto a sample plate; processing the sample by treating the sample with at least one of a solvent, a MALDI matrix chemical, water, and an acid, and mixtures thereof; and analyzing the sample using a diagnostic device. [Technical feature 35] 35. The method according to technical feature 34, wherein said EBA particles have at least one of microbial, viral, metabolic, lipid and proteomic biomarkers characteristic of respiratory disease. [Technical feature 36] 35. The method according to technical feature 34, wherein the processing step further comprises concentrating the sample by drying the sample using a suitable drying means. [Technical feature 37] 35. The method according to technical feature 34, wherein the diagnostic device comprises a MALDI-TOFMS. [Technical feature 38] said step of collecting using said sample capture component further comprising: flowing the output from the extracting step through a packed bed column to selectively capture the EBA particles; and extracting the EBA particles from the packed bed column using at least one of about 12.5% ​​acetic acid, about 70% isopropanol, about 5% TFA, about 5% formic acid, and about 10% HCl in a sample extraction system to generate a collection sample. [Technical feature 39] 35. The method according to technical feature 34, further comprising a step of digesting the collected sample to generate a peptide sample characteristic of EBA particles. [Technical feature 40] The packed bed column is made of resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4 39. The system according to technical feature 38, having solid particles having at least one of the nanoparticles. [Technical feature 41] 39. The system according to technical feature 38, wherein the packed bed column has resin beads having C18 functional groups on the surface. [Technical feature 42] 1. An autonomous method for diagnosing a respiratory disease in an individual using exhaled breath, comprising: The method comprises: instructing the individual to place a sample extraction component to extract EBA particles from the exhaled breath; initiating said predetermined number of breaths to expel EBA particles from the exhaled breath into the air stream that is supplied to the sampling component; flowing airborne EBA particles into the sample capture component while cooling the walls of the sample capture component and an interface tube fluidly connecting the sample capture component with a sample extraction component; generating collected samples; processing the collected sample, comprising treating the sample with at least one of a solvent, a MALDI matrix chemical, water, and an acid, and mixtures thereof; and analyzing the plated samples using a MALDI-TOFMS. [Technical feature 43] 43. The method according to technical feature 42, wherein the flow rate of air entering the sample capture component is between about 100 L / min and about 1000 L / min. [Technical feature 44] 43. The method according to technical feature 42, wherein the flow rate of air entering the sample capture component is between about 50 L / min and about 500 L / min. [Technical feature 45] 43. The method according to technical feature 42, wherein the collected sample volume is between about 100 microliters and about 1 ml. [Technical feature 46] The predetermined number of breathing maneuvers a. taking a deep breath to clear the individual's lungs; b. pausing for up to 10 seconds; c. performing an FVC inhalation; d. Take a deep breath and e. Repeating the above sequence up to 10 times. [Technical feature 47] The method according to technical feature 46, further comprising at least one of the steps of exhalation breathing, coughing, normal FVC breathing, speaking, and sneezing. [Technical Features 48] 43. The method according to technical feature 42, wherein the respiratory disease comprises at least one of tuberculosis, influenza, pneumonia, cancer, and a disease caused by a coronavirus. [Technical feature 49] 43. The method according to claim 42, wherein the predetermined number of exhalation maneuvers is determined by one or more sensors indicative of at least one of the volume of exhaled particles and the volume of exhaled air. [Technical Features 50] The sensor or sensors 2 The method according to technical feature 49, having at least one of a sensor, an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer. [Technical features 51] 1. A system for diagnosing respiratory illness caused by aerosolized viral and bacterial particles, comprising: The system is a sample capture component that collects a predetermined volume of airborne EBM particles in a stream of air provided to the sample capture component as a collected sample, the stream of air being between about 30 L / min and about 1000 L / min; a sample analysis subsystem fluidly connected to the sample capture component; The system is characterized in that the sample analysis subsystem includes a sample processing component that spots small amounts of the collected sample onto a sample plate and handles the collected sample on the sample plate, and a diagnostic device for analyzing the sample. [Technical features 52] The system according to technical feature 51, wherein the sample processing component has a fluid component for processing the sample using at least one of a solvent, a MALDI matrix chemical, water, and an acid, and mixtures thereof. [Technical features 53] The system described in technical feature 51 further comprises one or more sensors configured to be fluidly connected to the sample extraction component, and the output of the one or more sensors is used to calculate the total cumulative amount of exhaled aerosol particles entering the sample capture component. [Technical feature 54] The sensor or sensors 2 The system according to technical feature 53, having at least one of a sensor, an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer. [Technical features 55] The system of technical feature 51, wherein the predetermined volume of air is determined using the output of one or more sensors.

[0060] [References] 1. B. Bake, P. Larsson, G. Ljungkvist, E. Ljungstrom, and A-C Olin, "Exhaled particles and small airways," Respiratory Research (2019) 20:8. 2. Fennelly K.P., Martyny J.W., Fulton K.E., Orme FM., Cave D.M., et al. (2004) Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness. Am J Respir Crit Care Med 169: 604-609. 3. Dina Hashoul and Hossam Haick, "Sensors for detecting pulmonary diseases from exhaled breath," Eur. Respir. Rev. 2019; 28: 190011. 4. Hunt, J., "Exhaled breath condensate: An evolving tool for noninvasive evaluation of lung disease," J. Allergy Clin. Immunol. 2002; 110:28-34. 5. Maria D. King, Andrew R. McFarland, "Bioaerosol Sampling with a Wetted Wall Cyclone: Cell Culturability and DNA Integrity of Escherichia coli Bacteria," Aerosol Sci. Technol, 46:82-93, 2012. 6. James J. McDevitt, Petros Koutrakis, Stephen T. Ferguson, Jack M. Wolfson, M. Patricia Fabian, Marco Martins, Jovan Pantelic, and Donald K. Milton, "Development and Performance Evaluation of an Exhaled-Breath Bioaerosol Collector for Influenza Virus," Aerosol Sci. Technol. 2013 January 1; 47(4): 444-451. 7. Benjamin Patterson, Carl Morrow, Vinayak Singh, Atica Moosa, Melitta Gqada, Jeremy Woodward, Valerie Mizrahi, Wayne Bryden, Charles Call, Shwetak Patel, Digby Warner, Robin Wood, "Detection of Mycobacterium tuberculosis bacilli in bio-aerosols from untreated TB patients," Gates Open Research 2018, 1:11. 8. Wood R., Morrow C., Barry C.E., III, Bryden W.A., Call C.J., Hickey A.J., et al. : Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC). PLoS One. 2016; 11(1): e0146658. 9. Rachel C. Wood, Angelique K. Luabeya, Kris M. Weigel, Alicia K. Wilbur, Lisa Jones-Engel, Mark Hatherill, and Gerard A. Cangelosi, "Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients," Sci. Rep. 5, 8668 (2015). 10. Fatima B. Wurie, Stephen D. Lawn, Helen Booth, Pam Sonnenberg, Andrew C. Hayward, "Bioaerosol production by patients with tuberculosis during normal tidal breathing: implications for transmission risk," Thorax 2016; 71: 549-554. [Explanation of symbols]

[0061] 100 Diagnostic Systems 101 Sample Collection Subsystem 102 Sample Analysis Subsystem 104 Sample Extraction Component 105 patients / individuals Booth 106 107 Air Source 108 Sample Capture Components 109 Interface Tube 110 Sterile Components 111 Throat area 112 Sensors 113 Peltier Thermoelectric Cooling Device 115 EBA condensed liquid sample 116 Pump 117 Valve 119 HEPA Filter 120 Sample Processing Components 122 Diagnostic Equipment

Claims

1. 1. A system for collecting a condensed liquid sample containing exhaled breath aerosol (EBA) particles, comprising: a sampling component configured to receive the individual's face to sample exhaled breath aerosol (EBA) particles expelled from the individual during a predetermined number of breathing maneuvers; an air pump for providing a continuous flow of sheath air to the sampling component to transport the exhaled breath aerosol (EBA) particles through the sampling component; A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles, comprising: a sample capture component fluidly connected to the sample extraction component, the sample capture component including one or more coolers for cooling the sample capture component to form a condensed liquid sample containing exhaled aerosol (EBA) particles.

2. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 1, further comprising a HEPA filter disposed in fluid communication with the air pump to provide a continuous flow of filtered sheath air to the sample extraction component.

3. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 2, further comprising a humidifier configured to humidify the continuous flow of filtered sheath air before it enters the sample extraction component.

4. 2. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 1, wherein the sample extraction component comprises one or more of a CPR rescue mask, a CPAP mask, a ventilator mask, or a medical universal mouthpiece.

5. The system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 1 , wherein the sample extraction component includes a cone-shaped device configured to receive an individual's face.

6. 2. A system for collecting condensed liquid samples containing exhaled aerosol (EBA) particles as described in claim 1, wherein the air pump is configured to output air at a flow rate of 100 L / min ± 10% to 1000 L / min ± 10%.

7. A system for collecting a condensed liquid sample containing exhaled breath aerosol (EBA) particles as described in claim 1, further comprising a containment booth configured to receive the individual and isolate the individual's exhaled breath from ambient air, wherein the sample extraction component is fluidly connected to the sample capture component through a wall of the containment booth.

8. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 1, further comprising one or more sensors configured to be in fluid communication with the sample extraction component, the output of the one or more sensors being used to calculate a total cumulative amount of exhaled aerosol particles entering the sample capture component.

9. The one or more sensors ２ 9. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 8, comprising one or more of a sensor, an oxygen sensor, a humidity sensor, an optical particle size counter, an aerodynamic particle sizer, and a nephelometer.

10. 2. A system for collecting a condensed liquid sample containing exhaled aerosol (EBA) particles as described in claim 1, wherein the volume of the collected condensed liquid sample is between 100 microliters ± 10% and 1 microliter ± 10%.

11. 10. The system for collecting a condensed liquid sample containing exhaled breath aerosol (EBA) particles of claim 1, further comprising a sterilization component for sterilizing the sample capture component.

12. 12. The system for collecting condensed liquid samples containing exhaled aerosol (EBA) particles as described in claim 11, wherein the sterilization components include one or more of a nebulizer for spraying a disinfectant, one or more UV light sources for generating UV radiation, a steam generator, an ozone generator, a peroxide vapor generator, or combinations thereof.

13. 2. The system of claim 1, wherein the sample capture component comprises one or more of a cyclone, a wetted wall cyclone, one or more wet film impactors, or an impinger.

14. The system of claim 1 , wherein the sample capture component includes at least one virtual collision stage.

15. 2. The system for collecting a condensed liquid sample containing exhaled breath aerosol (EBA) particles as described in claim 1, further comprising an interface tube disposed between the sample extraction component and the sample capture component.

16. 16. The system for collecting a condensed liquid sample containing exhaled breath aerosol (EBA) particles as recited in claim 15, wherein the interface tube is fabricated from one or more of copper, or nickel-copper alloy 400.

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